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1 RESEARCH ON CHEMICAL COMPONENTS AND FUNCTIONAL PROPERTIES OF FISH PROTEIN ISOLATE (FPI) FROM BY-PRODUCTS OF Pangasius hypophthalmus Cao Xuan Thuy 1 , Tran Bich Lam 2 , Ha Thanh Toan 3 , Mac Xuan Hoa 4 1 Faculty of Food Technology, Hochiminh City University of Food Industry (HUFI). 2 Department of Food Technology, HCMC University of Technology -VNU 3 Biotechnology R&D Institute, Can Tho University 4 Faculty of Food Technology, Hochiminh City University of Food Industry (HUFI). ABSTRACT Currently, the utilization of Pangasius hypophthalmus by-products and processing them into high value added products will bring high economic efficiency for producers and reducing the amount of by-products cause environmental pollution. After hydrolysis of these by-products by enzyme Alcalase 2.4L, we get the fish protein isolates (FPI) that the major component is the peptides with different molecular weight. FPI have some different technology features that can be applied in food industry. This study focuses on examining the molecular weight of peptides in FPI; chemical components as well as functional properties of FPI. As a result, FPI contains the peptides with molecular weight from 3.4 kDa to 10 kDa (mainly in the range of 5kDa - 8kDa); chemical compositions: protein 90.8%, ash 4.3%, fat 0.76%, the remaining moisture. The nature and function properties of the FPI include: the ability to foam and emulsification. Foaming Index of the FPI is 0.77 at pH = 5.5, Emulsifying Activity Index (EAI) of the FPI is 206 at pH = 10 1. INTRODUCTION Fish protein isolate (FPI) is the product that contains mostly the peptides with small molecular weight - from 1kDa to around 10kDa (one might call peptone from fish). When hydrolysing Pangasius hypophthalmus by-product to obtain FPI, we can use endoprotease enzymes such as: Alcalase, Protamex, Flavozyme . Currently, in Vietnam, It is most popular to use Alcalase to hydrolyse Pangasius hypophthalmus by-products and the final products including: Fish Protein Concentrates (FPC), products output of fishmeal, fish oil… but these ones are raw products of low economic efficiency. The studies of FPI producing in Vietnam are still very modest. Up to now, we have finished studying the hydrolysis of Pangasius hypophthalmus products by enzyme Alcalase 2.4L in the optimum conditions (temperature, pH, rate of enzyme/substrate - E/S, hydrolysis time, the percentage of added water) to capture the hydrolysate containing small molecular weight peptides as the basis for the production of FPI. In other countries - especially the United States and Western Europe, FPI is derived from fish have been produced and have many important applications in the food processing industry. This article focuses on the research of molecular weight of peptides in the FPI; FPI chemical contents and initial examining of two functional properties (foaming, emulsification) of the FPI which is derived from Pangasius hypophthalmus by-products. Then giving its suggested applications in food industries in Vietnam 2. MATERIALS AND METHODS 2.1. Materials Fish protein solution obtained from the hydrolysis by-products of Pangasius hypophthalmus in optimum conditions (temperature, pH, rate of enzyme / substrate - E / S, hydrolysis time, the percentage of added water) will be concentrated, spray-dried to acquire FPI. FPI is divided into small unit (2-5 gram), cold storage at low temperature (0 0 C - 4 0 C) during the study. The control-sample proteins (such as casein, albumin .) using to compare the functional properties with the ones of FPI were purchased from EAC Co., or the Prestige Company from Denmark. 2 All chemical reagents used for the experiments were of analytical grade. 2.2. Methods The study of the molecular weight peptides in the hydrolysate/FPI: The hydrolyzate obtained after hydrolysis of Pangasius hypophthalmus by-products is brought to agarose gel electrophoresis (polysaccharide) to determine the molecular weight of the peptides. Then the hydrolysate is concentrated, spray-dried to collect FPI. The quantified study of the FPI’s chemical components: Lipid content was determined by Soxhlet method. Total Crud Nitrogen (NX6, 25) content was determined by Micro-Kjeldahl method. Determination of moisture content by standard AOAC. Determination of ash content by standard AOAC Determination of foaming possibility by method Tsumuraa Kazunobu (2004). Possibility of foaming is represented by the following formula: Determination of emulsifying ability by Kazunobu Tsumuraa method (2004). Emulsification was shown by emulsification index (EAI-Emulsifying Activity Index) as below formula: Where: T = 2.303 Delution factor = 2000 c = amount of protein in a unit volume (g / mL) Ф = volume of oil (0.25) The experimental data were processed by ANOVA analytical methods 3. RESULTS AND DISCUSSION 3.1. Determination the molecular weight of the peptides After hydrolysis of Pangasius hypophthalmus by-products by enzyme Alcalase 2.4L in optimal conditions, we use the sieving-filter (with a diameter of 0.2 mm sieve eyes) for seperating the hydrolysate and the solid. The hydrolysate is pre-treated as follows: Cooling to 4 0 C for 15 minutes to separate fat from the hydrolysate (when cooling, the fat will emerge to the surface of the hydrolysate); then centrifuging at a speed of 5000 rounds per minute (r.p.m) to separate the hydrolysis residues. Bringing the liquid obtained by centrifugation to gel agarose for electrophoresis. The electrophoresis results showed in figure 1. Entire volume of foam - Volume of water extracted Possibility of foaming = Initial volume 3 Figure 1. Results of electrophoresis of hydrolysate after centrifugation After hydrolysis, we found that the molecular weight of peptides is 3.4 kDa to 10 kDa, but almost between 5kDa-8kDa. In addition, during the hydrolysis, every 30 minutes we extract the hydrolysate, pre- treatment and provide electrophoresis to determine the molecular weight of peptides in hydrolysate. Here are the comparing results of molecular weights of peptides during hydrolysis: Figure 2. Results of electrophoresis of hydrolysate during the hydrolysis Where: S1: hydrolysate electrophoresis after 30 minutes of hydrolysis S2: hydrolysate electrophoresis after 60 minutes of hydrolysis S3: hydrolysate electrophoresis after 90 minutes of hydrolysis S4: hydrolysate electrophoresis after 120 minutes of hydrolysis S5: hydrolysate electrophoresis after 150 minutes of hydrolysis S6: hydrolysate electrophoresis after 180 minutes of hydrolysis 4 S7: hydrolysate electrophoresis after 210 minutes of hydrolysis SS: standard samples (standard marker proteins) Based on the results of electrophoresis, we found that: After 30 minutes of hydrolysis, peptides will form a long streak on the gel agarose; molecular weight of peptides is from 3.4kDa-100kDa. After 60 minutes of hydrolysis, the peptides were cleaved into small ones, they distributed between 5kDa and 30kDa, but most of them concentrated in the approximately from 10kDa to 25kDa. For S3 and S4 (from 90-120 minutes of hydrolysis), the proteins were separated into distinctly small segments because the hydrolysis was started to break down protein into smaller segments. After the period of 150-180 minutes, we found that the molecular weight of peptides concentrated between 5kDa-15kDa, mostly in the segments of 10kDa. After 210 minutes (S7) hydrolysis is completely, we found that the peptide molecular weight is from 3.4kDa to 10kDa, mainly concentrated in the range of 5kDa-8kDa. 3.2. Determining the chemical compositions of FPI In order to get the high yield as well as the desired quality of FPI, after centrifugation to separate the residues and hydrolysate, the hydrolysate needs to be concentrated (dry weight contents levels up to ≥ 10% comparing to total volume of hydrolysate) for making conveniences for spraying-dry. The hydrolysate is vacuum rotation concentrated in condition of temperature at 70 0 C; rotation speed at 42 r.p.m and the concentration time is 1 hour. After concentration, the amount of water to be separated from the sample is about 60%. After concentration, eliminating about 60% water from the hydrolysate and then spraying- dry in the conditions: Temperature T = 160 0 C; pressure: 3 bar; and input pumping speed n = 14 r/min (equivalent to the input flow at 32.5 ml/min.). The hydrolysate after vacuum concentration has the total dry weight content up to 10.15% comparing to total volume of hydrolysate. The FPI that obtained after spraying-dry had 93.29% dry weight content. So the FPI yield is calculated to be 55.14%. This result shows that there was loss of FPI due to the adhesion of FPI on the surface of drying device leads to low yield. The FPI received from the spraying-dry is analyzed for determining the FPI’s chemical compositions. Taking 15 samples and analyzing their chemical compositions, the results is as follows: (see Table 1 and Figure 3) Table 1. Chemical compositions FPI derived from Pangasius hypophthalmus by-products N 0 PROTEIN LIPIT ASH MOISTURE % % % % 1 91.03 0.93 4.95 3.09 2 90.08 0.57 4.78 4.57 3 89.98 0.52 4.32 5.18 4 89.99 0.31 3.98 5.72 5 92.01 0.73 3.69 3.57 6 92.57 0.95 4.01 2.47 7 91.05 0.70 4.31 3.94 8 91.07 0.59 4.32 4.02 9 90.21 0.98 4.51 4.30 10 90.09 0.83 4.76 4.32 11 91.03 0.92 3.57 4.48 12 90.19 0.59 4.53 4.69 13 90.05 0.86 4.74 4.35 14 91.89 0.94 4.02 3.15 5 15 92.00 0.91 4.00 3.09 1 00 92.00 0.91 4.00 3.09 AVERAGE 90.88 ± 2.10 0.76 ± 0.04 4.30 ± 0.21 4.06 ± 1.1 Figure 3. Chemical compositions FPI derived from Pangasius hypophthalmus by-products The protein content in FPI is relatively high (90.88%), low levels of fat (0.76%), ash is 4.3% and 4.06% of moisture. When compared these contents to FPI which derived from surimi of Pseudosciaena crocea (called Yellow coaker - one kind of marine fish) or FPI made from whole- body cod, we found that there is not a large differences betwen the chemical components of FPI from Pangasius hypophthalmus by-products and FPI from Yellow coake or cod. (see Table 2 and Figure 4). Table 2. Chemical compositions of FPIs from Pangasius hypophthalmus by-products, surimi of Pseudosciaena crocea, whole-body of cod. SOURCES PROTEIN (%) LIPID (%) ASH (%) MOISTURE (%) SUM (%) FPI from Pangasius hypophthalmus by-products 90.88 0.76 4.3 4.06 100 FPI from surimi of Pseudosciaena crocea 92.18 0.51 4.2 3.11 100 FPI from whole body of cod 91.04 0.32 4.01 4.63 100 6 Figure 4. Chemical compositions of FPIs from Pangasius hypophthalmus by-products, surimi of Pseudosciaena crocea, whole-body of cod 3.3. Determining the foaming ability of FPI To determine the foaming features of the FPI from Pangasius hypophthalmus by-products, we study and compare the forming ability of FPI from Pangasius hypophthalmus by-products with the foaming ability of two products: Protein Isolate derived from mushrooms (MPI) and albumin from egg. Dividing these FPIs and MPI into small units of 0.25 gram, dissolving in 25 ml of water. Adjusted pH = 4; 5.5; 7.0; 8.5; 10 (the pH values is basing on the study foaming features of albumin - by Douglas C. Montgomery, 2009) by HCl 2M. The solutions were then hit with a mixer in order to make foam at room temperature. After 30 seconds since finished the mixing, measuring the total volume in foaming phase (entire volume of foam), and the water volume that separated from total volume (volume of water extracted) and determining the foaming abilities of FPIs and MPI. The results were processed by ANOVA analytical method and presented in Table 3. Table 3. The results of the foam features of FPI from Pangasius hypophthalmus by-products, MPI and albumin. pH 4,0 5,5 7,0 8,5 10,0 Foaming ability of MPI 0,49±0,03 ac(*) 0,63±0,04 b(*) 0,53±0,03 c(*) 0,45±0,02 ad(*) 0,40±0,02 d(*) Foaming ability of FPI from Pangasius hypophthalmus by- products 0,77±0,03 a(*) 0,77±0,04 a(*) 0,75±0,02 a(*) 0,71±0,01 b(*) 0,69±0,01 b(*) 7 Foaming ability of Albumin 1,25±0,04 a(*) 1,10±0,06 b(*) 0,89±0,05 c(*) 1,27±0,02 a(*) 0,91±0,05 c(*) (*): the small exponential character: a, b, c, d showing the difference was significant or not significant in ANOVA analytical method. In case of contain the same character, the difference of foaming abilities is not significant; and the difference character shows the significant difference of foaming abilities) Figure 5. Foaming abilities FPI dirived from Pangasius hypophthalmus by-products, MPI (Mushroom Protein Isolate) and albumin From the results that show in figure 5, the foaming ability of FPI from Pangasius hypophthalmus by-products is better than the one of MPI, but much less than the one of albumin from egg. However, according to research by Nabil Souissi, Ali Bougatef, Yousra Triki-Ellouz and Moncef Nasr with the sardines substrate in low hydrolysis degree of <6%, the foaming ability of FPI from sardines is better than the one from MPI. The foaming formation of FPI (including FPI from Pangasius hypophthalmus by-product or MPI) were reduced when increasing pH, but the reduction was not too much. For albumin, the foaming ability reaches the highest level at pH 4 and pH 8.5, much higher than other pH values, and it gets the lowest value at pH 7 The foaming ability of FPI which derived from Pangasius hypophthalmus by-product reaches the peak at pH 5.5 and it is lower at other pH values. But differences were not significant (P <0.05) because the FPI weak foaming ability due to too small molecular weight of peptides in it. This may explain that the pH at this value is the iso-electric point (pI or pHi) of protein. At the pI, the molecules appear the electrostatic attractive forces to make the aggregation and increase the durability of the membranes around the air bubbles. At other pH values, the foaming ability is very low The foaming features of FPI from Pangasius hypophthalmus by-products is lower than the one of albumin can be explained that the small molecular weight of peptides inside FPI would interfere formation of solid membranes surrounding the air bubbles; and by the making of many hydrophilic peptides when the fish by-products hydrolysis get high efficiency. The hydrophilic peptides with unstable membranes will make the unstable foam. 3.4. Determining the emulsifying possibility of FPI To examine the emulsifying ability of FPI from Pangasius hypophthalmus by-products, we 8 make the emulsifier by taking 3 ml of protein (0.5 g/100mL) in McIlvaine buffer 35mmol/L (pH at 4; 5.5 and 7) and added 1 ml of soy oil. The mixture were homogenised by Ultrasonic Machine (5281 ultrasonic disperser from Kaijo Denki Co., Tokyo, Japan) for 2 minutes at 150W. Emulsifier is immediately diluted 2000 times (see 2.2.) with solution 1 g/L SDS and the turbidity was measured at a wavelength of 500 nm. Then determining the EAI (Emulsifying Activity Index). The emulsifying ability of the FPI from Pangasius hypophthalmus by-products was compared with the ones of casein and MPI (Mushrooms Protein Isolate). The results were processed by ANOVA analytical method , presented in Table 4 and Figure 6. Table 4. Emulsifying features of the FPI, MPI and Casein pH 4 7 10 EAI (Emulsifying Activity Index) of FPI from Pangasius hypophthalmus by-products 54,1±2,29 a(*) 55,8±2,12 a(*) 206±6,76 b(*) EAI (Emulsifying Activity Index) of MPI 64±3,63 a(*) 85,3±3,24 b(*) 213,5±7,71 c(*) EAI (Emulsifying Activity Index) of Casein 45,2±4,15 a(*) 117,6±7,84 b(*) 216,3±8,99 c(*) (*): the small exponential character: a, b, c showing the difference was significant or not significant in ANOVA analytical method. In case of contain the same character, the difference of EAI is not significant; and the difference character shows the significant difference of EAI) Figure 6. Emulsifying ability of FPI from Pangasius hypophthalmus by-products, MPI and Casein Figure 6 shows the differences of emulsifying ability among FPI from Pangasius hypophthalmus by-products, MPI and the control sample (casein). The results showed that the emulsifying abilities is significantly affected by pH (p <0.05). The emulsifying ability of the FPI from Pangasius hypophthalmus by-products is always lower than the MPI (Mushroom Protein Isolate) 9 The emulsifying ability of the FPI from Pangasius hypophthalmus by-products is also smaller than casein (except at pH 4, the emulsifying ability of the FPI of from Pangasius hypophthalmus by-products is higher than casein). At pH 4, the EAI of the FPI is 54.1 while the one of casein (control sample) is 45.2. This result can be explained by the influence of pH to the thrust forces of the polypeptide molecules. The results in figure 6 also show that at pH 10, the emulsification abilities of all samples are highest (P <0.05). Emulsification ability of FPI from Pangasius hypophthalmus by-products is low because this FPI contains the small peptides. These ones generally reduce the ability to create emulsions. In fact, a peptide is required a length of at least about 20 amino-acids for acting as a base to create emulsifying and make the surface distribution phases. At high pH value (pH = 10), the structure of the poly-peptide is stretched due to the same sign electricity on it. The same sign electricity could cause the repulsion and a better orientation of poly-peptides on the surface of distribution phases. This can lead to reveal the original hydrophilic and hydrophobic base/roots, which promote interaction of the surface distribution of oil in water (O/W) phase pH value is a major factor to be considered carefully when evaluating the emulsifying property. The emulsification index of the hydrolysis products at pH 10 is the highest and EAI (emulsification ability index) was 206 (P <0.05). No differences were found in the emulsification indexes of products (P <0.05) at pH 4 and pH 7. 4. CONCLUSION Researching on the peptides molecular weight, chemical compositions and some functional properties of FPI from Pangasius hypophthalmus by-products, we could conclude the results as follows: Molecular weight of the peptides in the FPI from Pangasius hypophthalmus by- products is from 3.4kDa - 10kDa, mainly about 5kDa-8kDa. The average chemical compositions of the FPI: protein 90.8%, ash 4.3%, fat 0.76%, the remaining moisture. Foaming index of the FPI from Pangasius hypophthalmus by-products is 0.77 at pH = 5.5 Index ability emulsification (EAI) of the FPI from Pangasius hypophthalmus by- products is 206 at pH = 10. We are continuing to study the technology properties of the peptides that have different molecular weight in FPI from Pangasius hypophthalmus by-products and then giving its suggested applications in food industries in Vietnam. 10 REFERENCE [1] Adler Nissen J.(1986). Enzymatic Hydrolysis of Protein in Food. Novo Nordisk [2] Amarowicz, R., & Shahidi, F. (1997). 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Protein composition isolated from a muscle source, isolated source. . 91.03 0.93 4.95 3.09 2 90.08 0.57 4.78 4.57 3 89.98 0. 52 4. 32 5.18 4 89.99 0.31 3.98 5. 72 5 92. 01 0.73 3.69 3.57 6 92. 57 0.95 4.01 2. 47 7 91.05 0.70 4.31. 0.59 4. 32 4. 02 9 90 .21 0.98 4.51 4.30 10 90.09 0.83 4.76 4. 32 11 91.03 0. 92 3.57 4.48 12 90.19 0.59 4.53 4.69 13 90.05 0.86 4.74 4.35 14 91.89 0.94 4. 02 3.15